obtaining wildtype and transgenic zebrafish larvae and wildtype whole larvae dissociation

עיבוד תאי מעיים מזחלי דגי זברה לריצוף RNA חד-תאי

The gastrointestinal (GI) tract is a complex system that plays a vital role in overall health and well-being. It is responsible for the digestion and absorption of nutrients, as well as the elimination of waste products1,2. The GI tract is composed of multiple cell types, including epithelial cells, smooth muscle cells, immune cells, and the enteric nervous system (ENS), which communicate closely together to regulate and maintain proper intestinal function3,4,5. Defects in the development of the GI tract can have far-reaching effects on various aspects such as nutrient absorption, microbiota composition, the gut-brain axis, and the ENS, leading to several enteric neuromuscular disorders, such as Hirschsprung disease and Chronic intestinal pseudo-obstruction6,7. These disorders are characterized by severe gut dysmotility caused by alterations in various key cells, such as the interstitial cells of Cajal, smooth muscle cells, and the ENS6,8,9. However, the molecular mechanisms underlying GI development and dysfunction are still poorly understood.
The zebrafish is a valuable model organism for studying GI development and dysfunction due to its rapid embryonic development, transparency during embryonic and larval stages, and genetic tractability10,11,12,13,14. Numerous transgenic zebrafish lines expressing fluorescent proteins are available. An example of such a line is the tg(phox2bb:GFP) zebrafish, commonly used to study the ENS, as all phox2bb+ cells, including enteric neurons, are labeled15,16. Here, using the tg(phox2bb:GFP) zebrafish line, we present a method for intestinal isolation of 5 days post fertilization (dpf) larvae for single-cell RNA sequencing (scRNA-seq) analysis (Figure 1).

מחבר זרקור: בידוד וניתוח תאי מעיים של זחלי דגי זברה לחקר היבטים שעתוק של התפתחות מערכת העיכול 

בידוד מעיים מזחלי דגי זברה לריצוף RNA חד-תאי

Our research aims to investigate the transcriptomic changes occurring during gastrointestinal development and dysfunction in zebrafish larvae. Here, we provide a new protocol that allows isolation of different cell types within the zebrafish intestine, which has potential applications in understanding gastrointestinal health and disease. In recent years, there has been growing interest in exploring the interaction between the enteric nervous system and other intestinal cell types during development and disease.Understanding these complex dynamics using advanced techniques like single-cell RNA sequencing, holds the potential to reveal critical insights into enteric neuromyopathies, leading to more precise diagnostics and treatments. Using this protocol, we successfully identified various intestinal cell types, including epithelial, stromal, bloods, muscle, immune cells, and even enteric neurons and glia, which have not been captured before at this amount using similar approaches. Thus, we provide a reliable technique to study the gastrointestinal composition during development and dysfunction.

Watch this Scientific Journal Video about Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation at JoVE.com

Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation  

השגת זחלי זברה מהונדסת וטרנסגנית ודיסוציאציה של זחלים שלמים מסוג WildType

התחילו בהשגת ביצי דגי הבר ודגי הזברה הטרנסגניים ממערך הרבייה המתאים. קחו צלחת פטרי בקוטר 15 ס"מ המכילה מדיום E3 חוצץ HEPES או E3. ולאסוף את הכמות המתאימה של ביצים מופרות ממערך הרבייה הרצוי של דגי זברה. לדגור על הביציות המופרות ב-E3 בטמפרטורה של 28 מעלות צלזיוס עם מחזור אור וחושך מתאים.ביום אחד לאחר ההפריה או dpf, להסיר את כל הביציות לא מופרות ולתת אלה מופרות להתפתח עד חמש dpf. בחר את זחלי דגי הזברה הטרנסגניים ב- DPF אחד. לפני שממשיכים בדיסוציאציה של זחלים שלמים, מרדימים את חמשת זחלי הבר מסוג dpf באמצעות 0.016% טריקאין, ומעבירים 30 זחלים מורדמים לצלחת פטרי המכילה מיליליטר אחד של תמיסת טריפסין-EDTA 10x.לאחר מכן, באמצעות סכין גילוח, קוצצים את הזחלים לחתיכות קטנות בצלחת הפטרי. מעבירים את הזחלים הקצוצים לתוך צינור מיקרו צנטריפוגה עם שני מיליליטר של תמיסת טריפסין-EDTA 10x, ומשאירים את הצינור על קרח למשך שלוש שעות. פיפטה את התוכן למעלה ולמטה עם פיפטה P1000 כל שעה כדי לעורר דיסוציאציה.

obtaining-wildtype-transgenic-zebrafish-larvae-wildtype-whole-larvae

Research

JoVE Journal

Developmental Biology

Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation

115 Views.  Erasmus University Medical Center - Sophia Children's Hospital. התחילו בהשגת ביצי דגי הבר ודגי הזברה הטרנסגניים ממערך הרבייה המתאים. קחו צלחת פטרי בקוטר 15 ס"מ המכילה מדיום E3 חוצץ HEPES או E3. ולאסוף את הכמות המתאימה של ביצים מופרות ממערך הרבייה הרצוי של דגי זברה. לדגור על הביציות המופרות ב-E3 בטמפרטורה של 28 מעלות צלזיוס עם מחזור אור וחושך מתאי...

Video: Obtaining Wildtype and Transgenic Zebrafish Larvae and WildType Whole Larvae Dissociation  

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

Begin by obtaining the wildtype and transgenic zebrafish eggs from the respective breeding setup. Take a 15-centimeter Petri dish containing HEPES-buffered E3 medium or E3.And collect the appropriate amount of fertilized eggs from the desired zebrafish breeding setup. Incubate the fertilized eggs in E3 at 28 degrees Celsius with an appropriate light and dark cycle.At one days post fertilization or dpf, remove any unfertilized eggs and let the fertilized ones develop until five dpf. Select the transgenic zebrafish larvae at one dpf. Before proceeding for whole larvae dissociation, anesthetize the five dpf wildtype larvae using 0.016%Tricaine, and transfer 30 anesthetized larvae into a Petri dish containing one milliliter of 10x trypsin-EDTA solution.Then, using a razor blade, chop the larvae into small pieces in the Petri dish. Transfer the chopped larvae into a micro centrifuge tube with two milliliters of 10x trypsin-EDTA solution, and leave the tube on ice for three hours. Pipette the contents up and down with a P1000 pipette every hour to stimulate dissociation.

Isolation and Dissociation of Gut from Zebrafish Larvae for FACS Enrichment of Gut Cells

To begin, prepare a 1.8%agarose plate with HEPES Buffered E3 medium or E3.While working under a dissection microscope, place six to 10 anesthetized larvae in a row on the agarose plate. Put one insect pin on the head of each larvae. Then remove the remaining E3 from the plate using a tissue.Using another insect pin, isolate the intestine from a larvae without disturbing any other organs. Ensure proper removal of yolk. Once isolated, inspect the intestine and remove all non-intestinal material, such as skin, fat, or liver.Use tweezers to collect the intestine and transfer it to PBS containing 10%FCS in a microcentrifuge tube placed on ice. Immediately after the dissection of all intestines, centrifuge the microcentrifuge tube at 13, 800 g for 30 seconds. Remove the supernatant, leaving about 100 microliters in the tube to prevent the intestines from drying out.Add 500 microliters of papain solution to the intestines in the tube. Activate the papain by adding 2.5 microliters of one molar cysteine. Then incubate the tube containing the intestines in a water bath at 37 degrees Celsius for 10 minutes.Pipette the contents up and down halfway after five minutes to stimulate enzymatic tissue digestion. Transfer the dissociated cells into a fluorescence-activated cell sorting, or FACS, tube through a pre-wedded 35-micron cell strainer. Wash the strainer several times by adding 0.5 milliliters of PBS containing 10%FCS to a total wash volume of two milliliters.Centrifuge the collected filtrate at 700 g for five minutes at 4 degrees Celsius, and remove the supernatant. Add one microgram per milliliter DAPI to 300 microliters of PBS containing 10%FCS. Add this mixture to the pellet and resuspend to label the dead cells.Then incubate for five minutes on ice to allow their exclusion during subsequent FACS analysis.

בידוד ודיסוציאציה של המעי מזחלי דגי זברה להעשרת FACS של תאי מעיים

FACS Enrichment of Zebrafish Gut Cell Suspension for scRNAseq

To perform fluorescence-activated cell sorting, or FACS, on the gut cells isolated from zebrafish larvae, use the wild-type whole larvae sample to set the gates of the sorted cell population by recording 3, 000 cells on the flow cytometer. Use a 100-micron nozzle and set Sort Precision to 4-Way Purity. Put the collection tube containing 200 microliters of PBS with 5%fetal calf serum in the fax machine.Load the cell suspension prepared from the isolated guts, and after excluding doublets in dead cells, sort live or DAPI-negative single cells into the collection tube. Keep the collection tube on ice after cell sorting, employing a viability check with trypan blue. Count the number of cells in the suspension with a hemocytometer.The cells are now ready to be processed for single-cell RNA sequencing. Use approximately 20, 000 cells per sample.